ABSTRACT Our long-term goal is to develop novel antitumor therapies to treat cancer with elevated (> 100-fold) levels of NAD(P)H:quinone oxidoreductase 1 (NQO1). ?-Lapachone (?-lap), an NQO1 bioactivatable drug, selectively targets NQO1+ tumors and is activated by NQO1 to generate reactive oxygen species (ROS), leading to extensiveDNAdamageandPARP1-driventumorprogrammednecrosis.Ourpreliminarystudiesdemonstrate thatbothneutrophil-mediatedinnateimmunityandCD8-mediatedadaptiveimmunityarerequiredforantitumor efficacy of ?-lap in vivo. Furthermore,ourprevious studies reveal thattargeting NQO1 potently triggers innate sensing within tumor microenvironment (TME) that synergizes with immunotherapy to overcome adaptive resistance. Our objective here is to define and delineate the mechanism(s) of tumor-specific ROS and DNA damageinducedby?-lapthatstimulatesantitumorimmunity,anddeterminehow?-lapsynergizeswithimmune checkpointblockadetherapy.Ourcentralhypothesisisthat(i)?-laptreatmenttriggersimmunogeniccelldeath (ICD)andinducesdamage-associatedmolecularpatterns(DAMPs)release;?(ii)phagocytes/antigen-presenting cells (APCs) recruitment promotes cross-priming of cytotoxic T cells (CTLs) for suppression of tumor by increasingantigen/DNAuptakeandtypeIinterferons(IFNs)production;?and(iii)upregulatedPD-L1withinTME contributes to tumor relapse and provides therapeutic window for combination therapy of ?-lap with immune checkpoint blockade. We propose the following Specific Aims. AIM 1: Elucidate the mechanism of ?-lap- triggeredICDforinnateimmunesensing.Ourworkinghypothesisisthat?-laptriggersICDforinnatesensing viathereleaseofDAMPs.Wewillassesstheabilityof?-laptostimulatetumorICDinvivobytherapeuticvaccine assay.Wewillalsodeterminewhichtype(s)oftumorDNA(genomicormitochondrial)isthemajorsource(s)of IFNsproductionafter?-laptreatment.AIM2:Definehowtumorcellsandimmunecellscross-talkoccurs in?-lap-inducedantitumorimmunity.Ourworkinghypothesisisthat?-lap-inducedneutrophilscross-primeT cellsdirectlyorinteractwithDCs/macrophagestoprimeTcells.Wewilldeterminetheeffectsof?-laptreatment on cGAS/STING/IFNs involved in T cells cross-priming. AIM 3: Determine the mechanism by which ?-lap synergizes with immune checkpoint blockade therapy to efficaciously kill NQO1+ tumors. Our working hypothesisisthatincreasedPD-L1withinTMEcontributestotumorrelapseoflargetumorsafterinitialresponses to ?-lap. We will determine in whichtype(s) of cells PD-L1expression isupregulated within TME. We will also determinewhichtype(s)ofPD-L1-expressingcellsisessentialforthesynergisticeffectinmicewithconditional deficienciesof PD-L1 ontumors, DCs or macrophages. IMPACT: These studies willdelineate the role of ?-lap intumor-selectiveinnatesensingthatleadstoTcell-dependenttumorcontrol.Further,wewillelucidatehow?- lap overcomes adaptive resistance to anti-PD-L1 therapy. This research will provide novel evidence for new combinationtherapyforNQO1+solidtumorsandbroadentheclinicalutilityofimmunecheckpointinhibitors.